The human immunodeficiency virus type 1 (HIV) is the causative agent of the AIDS pandemic. In the absence of a vaccine, identification of targets for development of agents directed against the rapidly emerging resistant variants is critical. The virus must be released from infected cells to spread infection and for this process it exploits cellular proteins. The objective of this projet is to identify key proteins and determine how they facilitate assembly and release. Previous studies established the role of Tsg101 and ESCRT (endosomal sorting complexes required for transport) machinery in trafficking of the viral proteins to budding sites on the plasma membrane. Since this machinery functions to sort and deliver cell surface receptors whose signaling is no longer required to lysosomal or proteasomal compartments in the cell interior for degradation, understanding how the virus circumvents this fate will provide information for formulation of novel anti-viral strategies. Preliminary data derived through proteomic, biochemical and cell biological analyses conducted in the previous funding period indicates that the structural precursor polyprotein, Gag, which is the only viral gene product required for formation and release of virus-like particles, directs the recruitment of calcium signaling machinery for this purpose. We propose to identify the determinants in Gag that are required for recruitment (Specific Aim 1), define the contributions of the recruited cellular proteins (Specific Aim 2) and characterize their physical association to the viral protein and each other (Specific Aim 3). Chimeric Gag proteins with domains derived from HIV- 1 Gag, whose budding is dependent on the calcium machinery and avian sarcoma virus Gag, whose release is not, will be used to identify the key determinants. This approach will be complemented by mutational analysis of specific residues in the identified domains. Functional contributions of the recruited proteins in model cells (e.g., HeLa), T cells and macrophages will be investigated through siRNA-mediated depletion and replacement studies. Physical associations or proximity will be assessed using bimolecular fluorescence complementation, immunoprecipitation, immuno-electron microscopy and biotinylation assays. Direct interactions will be checked using protein- protein interaction assays. These studies will define the mechanism underlying viral exploitation of the ESCRT cellular machinery and provide new strategies for anti-viral drug development by identifying required cellular proteins and events that are potential targets for pharmaceutical interventions. PUBLIC HEALTH RELEVANCE: Although HIV-1 is still the cause of the world-wide AIDS pandemic and no vaccine is available, infection is no longer a death sentence thanks to interventions targeted at viral gene products, which are now being used as a functional cure approach. Nevertheless, major barriers to a cure persist because drug-resistant variants emerge rapidly. Discovery of new anti-viral targets is thus a continuing and critical priority. A strategy that should permit us to circumvent the lattr problem is development of anti-viral agents that target cellular proteins such as Tsg101 and endocytic machinery factors that play key roles in virus production.